c-di-GMP inhibits the DNA binding activity of H-NS in Salmonella

Cyclic di-GMP (c-di-GMP) is a second messenger that transduces extracellular stimuli into cellular responses and regulates various biological processes in bacteria. H-NS is a global regulatory protein that represses expression of many genes, but how H-NS activity is modulated by environmental signals remains largely unclear. Here, we show that high intracellular c-di-GMP levels, induced by environmental cues, relieve H-NS-mediated transcriptional silencing in Salmonella enterica serovar Typhimurium. We find that c-di-GMP binds to the H-NS protein to inhibit its binding to DNA, thus derepressing genes silenced by H-NS. However, c-di-GMP is unable to displace H-NS from DNA. In addition, a K107A mutation in H-NS abolishes response to c-di-GMP but leaves its DNA binding activity unaffected in vivo. Our results thus suggest a mechanism by which H-NS acts as an environment-sensing regulator in Gram-negative bacteria.

A second gap arises because the authors do not seem to address the presence in Salmonella of StpA, a closely-related paralogue of H-NS, that is encoded by the chromosomally-located gene, stpA.Given the wide sweep of H-NS-like proteins that the authors have considered in their analysis, it seems strange that they have not mentioned this second H-NS-like protein in Salmonella.Of the five amino acids in Salmonella H-NS implicated in c-di-GMP binding that are highlighted in yellow in Given the ability of StpA to form heterodimers with H-NS, readers will be curious to know if c-di-GMP can influence the binding of StpA to DNA, either as an StpA homodimer or as a heterodimer with H-NS.The heterodimer situation becomes even more complicated in Salmonella clinical isolates that have large plasmids expressing additional H-NS-like proteins (PMID: 20444106).
There is considerable overlap between the regulons of genes controlled by StpA and H-NS and StpA has a key role in controlling the production of RpoS, a sigma factor whose gene can acquire compensatory mutations in hns knockout mutants (PMID: 19843227, PMID: 26631971).Indeed, the stpA gene is also prone to the acquisition of compensatory lesions when hns is disrupted (PMID: 25375226).Before branching out to a discussion of H-NS-like proteins in other bacterial species, perhaps the authors could make an assessment of StpA in Salmonella?
Lines 168-170: In Salmonella, the Fur protein is a negative regulator of hns transcription (PMID: 21075923, PMID: 22017966).The authors will need to adjust their description of the roles of the Fur and H-NS proteins in the control of T6SS gene expression to take into account this Fur-hns direct regulatory relationship.Lines 179-182: "Despite the lethality of the hns null mutation in S. Typhimurium strain ATCC 14028s, S. Typhimurium strain LT2 has been shown to tolerate the hns mutation (15,26).Consistently, an hns deletion mutation was successfully constructed by the CRISPR-Cas9 system in strain SL1344."Knockout mutations in the Salmonella hns gene are tolerated when compensatory mutations arise either in the rpoS gene or the phoPQ operon.Strain LT2 is already defective in expressing full levels of RpoS (the stress and stationary phase sigma factor of RNA polymerase) so it tolerates inactivation of hns because, genetically, it is 'pre-compensated'.This is not the case with strain SL1344, used by the authors of the present study.Here, it is likely that the authors have isolated an SL1344 hns mutant that has compensatory lesions in either the phoPQ operon or the rpoS gene.This should be investigated because, if compensatory mutations are present, their data interpretation must be expanded to take into account the effects of these additional mutations.Both RpoS and PhoPQ exert widespread influence on the expression of Salmonella virulence genes, including many of the genes that the authors study in the present investigation.
It should perhaps be mentioned that the K107A mutant H-NS can still be displaced from DNA by other mechanisms (e.g. the intervention of anti-repressor proteins).These mechanisms have been described in numerous bacterial systems (PMID: 18757787).

Reviewer #3 (Remarks to the Author):
This paper looks at the connection between the signaling molecule c-di-GMP and the DNA-binding, silencing factor H-NS in Salmonella.The authors argue that (i) c-di-GMP directly binds H-NS, preventing H-NS from binding DNA, (ii) high c-di-GMP levels lead to de-repression of H-NSsilenced genes, (iii) de-repression of H-NS-silenced genes provides a fitness advantage because of increased Type VI secretion, and (iv) the functionally analogous MvaT protein from Pseudomonas is also bound and inhibited by c-di-GMP.These conclusions have major implications for our understanding of the role of c-di-GMP, and for how H-NS activity is modulated.The work is generally very thorough, and I have only one suggested experiment that would add an important control: -The authors should use ChIP-qPCR to test whether K107A H-NS binding is affected either by bile salts (or by a more direct modulation of c-di-GMP levels).
Additional comments: 1.I recommend moving Supplementary Figure 1 into Figure 1. 2. Change "Relative gene expression" labels on rtPCR graphs to "Relative RNA level".3. Data from Supplementary Figures 11 and 13 showing that the K107A H-NS mutant is unaffected by c-di-GMP are key to the main conclusions of the paper.I recommend moving these to the main figures.4.Many strains of LT2 are avirulent.Can the authors comment on the genotype of the strain they used? 5. Are there RNA-seq data from Salmonella or Escherichia coli with altered c-di-GMP levels?If so, the authors could use those data as an independent test of their model, since H-NS-repressed genes are well characterized in these species.6. Can the authors comment on why increased c-di-GMP levels in the two tested Pseudomonas species has the same phenotype as deleting mvaT, despite the fact that MvaU would be expected to contribute to repression?Related to this, the authors should at least mention MvaU.7. Deletion of hns is lethal in other Salmonella strains and some other species, and leads to a growth defect in E. coli.Can the authors speculate on the impact of raised c-di-GMP levels on bacterial fitness, since their model indicates that high c-di-GMP levels would derepress all H-NSsilenced genes?
Reviewer #4 (Remarks to the Author): c-di-GMP is a widespread secondary messenger in bacteria, which regulates numerous different processes, e.g.related to the planktonic/biofilm life style, motility, pathogenicity and more.Over the last decades a number of different effector proteins have been identified that bind and thereby respond to c-di-GMP.In this study, the authors convincingly demonstrate that the highly conserved histone-like nucleoid structuring protein H-NS is, in fact, a c-di-GMP-binding regulator in Salmonella Enterica.The authors present in vitro and in vivo approaches that demonstrate direct c-di-GMP binding to H-NS and provide evidence that the c-di-GMP-binding site partly overlaps with the DNA-binding site of H-NS, so that DNA interaction of the protein is abrogated upon rising levels of c-di-GMP.In S. Typhimurium they show by RNAseq approaches that a number of factors are regulated by this mechanism, among them activation of a type VI secrection system (T6SS), which occurs upon exposure of the cells to signals such as bile acids.By mouse models they show that, in fact, the signaling cascade from bile acids to T6SS activiation likely functions via c-di-GMP and H-NS and provides an advantage in successful colonization of the host gut.They also present evidence that H-NS in other gammaproteobacteria such as Vibrio parahaemolyticus, Pseudomonas aeruginosa and P. putida binds and responds to c-di-GMP binding, so that this mechanism is likely highly conserved.
The major and highly relevant finding that highly conversed H-NS serves as a c-di-GMP-binding regulator is convincing as it is supported by the results presented by the authors.The number of high-quality experiments is impressive and the manuscript is very well structured.I have only some minor comments with respect to this study.58, ‚RsmA acts as …'; this passage does not really fit there and should be moved to somewhere else or could be omitted completely.
153 and throughout manuscript, ‚significantly' could also be true to rather small differences.I suggest to at least sometimes mention the factor of changes (e.g.double, six-fold, ..) where apprpriate.
173, Fig. 2a and throughout manuscript: There are many bar diagrams that provide complex sets of data.To make it a little bit easier for the reader, the authors may consider to add more information to the figure panels, e.g., already indicate in the panel which strain is used.233, please state that alanine substitutions were introduced.234, and general, it would be nice to see a presentation of the protein purifications to visualize the quality of the purification.It would also be nice if, for example, a SEC elution profile for the substitution variants could be presented to demonstrate that the structure of the mutated protein is preserved.

259, is FLAG-H-NS functional?
355, I would ask the authors to add a little bit of explanation here -who ist competing and under which conditions.389, strongly, better indicate a factor here (see above) 390, Fig. S18 shows growth state-dependent, that should be mentioned here.456, please name the conserved residue Just as a final small remark: in basically all figures, axis labeling or the insets are sometimes really small and hard to make out in the PDF for reviewing.

Response: NT cWP]Z cWT aTeXTfTa
Fig 8c, two are not present in StpA.The tyrosine is replaced by phenylalanine and the valine by threonine.In the following alignment, the five amino acids are bracketed by spaces on either side to facilitate identification: H-NS(St) 91-AARPAKYS Y V D ENGET K TWTGQGR T-115 StpA(St) 91-QPRPAKYR F T D FNGEE K TWTGQGR T-115 Lysine K107 is present in both proteins.